Download Na /Ca2+ Exchanger Maintains Ionic Homeostasis in the

Naⴙ/Ca2ⴙ Exchanger Maintains Ionic Homeostasis in the
Peri-Infarct Area
Anna Tortiglione, PhD; Barbara Picconi, PhD; Ilaria Barone, PhD; Diego Centonze, MD;
Silvia Rossi, MD; Cinzia Costa, MD; Massimiliano Di Filippo, MD; Alessandro Tozzi, PhD;
Michela Tantucci, PhD; Giorgio Bernardi, MD; Lucio Annunziato, MD; Paolo Calabresi, MD
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Background and Purpose—A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both
Na⫹ and Ca2⫹ ions, which results in subsequent cell death. The plasma membrane Na⫹/Ca2⫹ exchanger (NCX), regulates
the distribution of these ions acting either in the forward mode or in its reverse mode and it can play a critical role in
brain ischemia. However, it is unclear whether the activity of NCX leads to detrimental or beneficial effects.
Methods—Extracellular field potentials and whole-cell patch clamp recordings were obtained from rat corticostriatal
brain-slice preparations in the peri-infarct area 24 hours after the permanent middle cerebral artery occlusion. Ischemia
was induced in rats by permanents middle cerebral artery occlusion.
Results—Bepridil, an inhibitor of NCX, reduced in a concentration-dependent manner (IC50⫽68 ␮mol/L) the field
potential amplitude recorded from the peri-infarct area of corticostriatal slices. Conversely, no change was observed in
sham-operated animals. The effect of bepridil was mimicked by 5-(N-4-chlorobenzyl)-2⬘,4⬘-dimethylbenzamil (CBDMB) (IC50⫽6 ␮mol/L), a more selective inhibitor of NCX. In whole-cell patch clamp experiments, bepridil and
CB-DMB caused an inward current in spiny neurons recorded from the peri-infarct area but not in the same cells
recorded from controls. Interestingly, cholinergic interneurons recorded from the striatal peri-infarct area did not
develop an inward current after the application of NCX inhibitors, suggesting that the electrophysiological alterations
induced by NCX inhibition are cell-type specific. Bepridil and CB-DMB also induced a suppression of excitatory
synaptic currents in most of spiny neurons recorded from the peri-infarct area. This effect was not coupled to a
significant change of paired-pulse facilitation suggesting a postsynaptic site of action.
Conclusions—Our data indicate that NCX plays a critical role in the maintenance of ionic homeostasis in the peri-infarct
area. (Stroke. 2007;38:1614-1620.)
Key Words: electrophysiology 䡲 field potential 䡲 ischemia 䡲 Na⫹/Ca2⫹ exchanger
䡲 permanent middle cerebral artery occlusion 䡲 striatum
n neuronal cells, the Na⫹/Ca2⫹ exchanger (NCX) plays a
fundamental role in controlling Na⫹ and Ca2⫹ homeostasis.1
Depending on the intracellular concentrations of Ca2⫹ and Na⫹,
NCX can operate either in the forward mode, coupling the
extrusion of Ca2⫹ to the influx of Na⫹ ions, or in the reverse
mode, mediating the extrusion of Na⫹ and the influx of the Ca2⫹
ions.1 This feature of NCX is of critical importance considering
that changes in the intracellular concentrations of Na⫹ and Ca2⫹
ions are known to occur in pathophysiologic states, such as brain
ischemia.2–9
Moreover, a prominent feature of cerebral ischemia is the
excessive extracellular accumulation of glutamate and subsequent impairment of intracellular Na⫹ and Ca2⫹ homeostasis,
I
which results in subsequent cell death.3,10,11 Thus, by controlling intracellular homeostasis of these 2 ions, NCX may play
a pivotal role in the events leading to ischemic damage.
Reduction of the energy supply leads to a cascade of events
including depolarization, influx of Na⫹, and the subsequent
reverse operation of the membrane protein that ultimately
terminates in intracellular Ca2⫹ overload and irreversible
neuronal injury.2,12
Conflicting reports have been published on the role played
by NCX during brain ischemia. The inhibition of NCX
prevents the activation of phospholipases that occurs after an
ischemic insult of the brain.13 Similarly, activation of NCX in
its reverse mode plays an important role in cellular Ca2⫹
Received November 24, 2006; accepted December 14, 2006.
From Fondazione Santa Lucia IRCCS (A.T., A.Tozzi, B.P., C.C., D.C., G.B., I.B., P.C.), CERC, Rome, Italy; Clinica Neurologica (D.C., G.B., I.B.,
S.R.), Dip di Neuroscienze, Università di Roma Tor Vergata, Rome, Italy; Division of Pharmacology (A.T., L.A.), Department of Neuroscience, School
of Medicine, “Federico II” University of Naples, Naples, Italy; Clinica Neurologica (A.Tozzi, C.C., M.D.F., M.T., P.C.), Dip Specialità MedicoChirurgiche, Università di Perugia, Perugia, Italy.
A.T. and B.P. contributed equally to this work and both should be considered first authors.
Correspondence to Professor Paolo Calabresi, Clinica Neurologica, Facoltà di Medicina e Chirurgia, Università degli Studi di Perugia, Ospedale
Silvestrini, S.Andrea delle Fratte, 06156 Perugia, Italy. E-mail [email protected]
© 2007 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
DOI: 10.1161/STROKEAHA.106.478644
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Tortiglione et al
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overload and irreversible damage after energy deprivation.13–16 Conversely, by using in vitro and in vivo models of
anoxia and ischemia, it has been demonstrated that the
stimulation of NCX activity may help neurons to survive,
whereas its pharmacological blockade can compromise their
survival.6,7,17,18 Accordingly, we found that the activation of
the NCX exerts a protective role during the early phase of
oxygen and glucose deprivation in striatal neurons.19
Considering this controversial issue, we have analyzed the
effect of the pharmacological inhibition of NCX on the
physiological properties of neurons recorded from the striatum, a structure that is highly vulnerable to ischemic insults.4,5,20,21 The mRNA and proteins of the 3 isoforms NCX1,
NCX2, and NCX3 are widely expressed in the striatum.9,22
However, their exact location on specific striatal neuronal
subpopulations as well as their role in cell-type–specific postischemic striatal damage has been not yet analyzed. Thus, we have
recorded the physiological activity of striatal neurons in the
peri-infarct area after permanent middle cerebral artery occlusion and their electrical response to the pharmacological inhibition of NCX.
In the peri-infarct area, we have also studied the possible
differential sensitivity to NCX inhibition of striatal spiny
neurons, a neuronal subtype that is highly sensitive to
ischemia and energy deprivation, versus striatal cholinergic
interneurons, that have been reported to be resistant to these
pathological conditions.4,5,20,21
Materials and Methods
Animals
Sprague-Dawley male rats (250 to 270 grams; Charles River, Lecco,
Italy) were used. All the experiments were conducted in conformity
with the European Communities Council Directive of November
1986 (86/609/EEC).
Permanent Middle Cerebral Artery Occlusion,
Monitoring of Regional Cerebral Blood Flow, and
Infarct Size Analysis
Methods for permanent middle cerebral artery occlusion have been
previously described.23,24 Regional cerebral blood flow was monitored in the ipsilateral parietal cortex to the occluded middle cerebral
artery using laser Doppler flowmeter (Periflux System 5000), provided with PC software (PeriSoft PSW 2.0) used to record the blood
flow.25
For the staining of infarct size analysis, 2,3,5-triphenyltetrazolium
chloride was used as previously described.24 The results are given as
infarcted volume, adjusted for edema index, as the percentage of the
whole ipsilateral cerebral hemisphere.26
Electrophysiology and Identification of the
Peri-Infarct Area
Preparation and maintenance of rat corticostriatal slices (270 to
300 ␮m) have been previously described.19,20,27 Slices were kept in
artificial cerebrospinal fluid (34° C, O2/CO2) whose composition was
(in mmol/L): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4
CaCl2, 11 glucose, and 25 NaHCO3. For synaptic stimulation, bipolar
electrodes were used, located in the white matter to activate
corticostriatal fibers.
Electrophysiological electrodes for extracellular recording (15 to
20 mol/L⍀) were filled with 2 mol/L NaCl.
The peri-infarct area was defined through electrophysiological
criteria.28 Recording electrodes were initially positioned within the
striatum. The stimulating electrode, constantly held 0.3 mm apart,
Naⴙ/Ca2ⴙ Exchanger and Ischemia
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was progressively placed in up to 8 distinct positions around the
recording electrode (360°) to try to elicit extracellular field potentials. We assumed we were in the ischemic core when no reliable
field potential (negative deflection from the baseline of at least 0.2
mV) was evocable despite the progressive increase of the stimulation
intensity. From the “core” position, the electrodes were progressively
(0.5 mm) moved along a single line toward the periphery of the
striatum, with the stimulating electrode preceding the recording one.
When the field potential appeared, we considered we were in the
peri-infarct area. In the “ischemic core” it was not possible to obtain
single cell recordings by using whole-cell patch clamp. Conversely,
in the peri-infarct area this technique allowed to obtain reliable
electrophysiological recordings. This was confirmed analyzing the
extension of the ischemic core after 2,3,5-triphenyltetrazolium chloride staining of the slices obtained immediately before and after the
one used for the electrophysiological experiments.
Whole-Cell Patch Clamp Recordings
Whole-cell patch clamp recordings and identification of individual
neurons were performed in situ using a differential interference
contrast (Nomarski) optical system as previously described.29 Striatal spiny neurons were clamped at ⫺80 mV, whereas striatal aspiny
interneurons were clamped at ⫺60 mV, close to their respective
resting membrane potentials. All the experiments were performed in
the presence of 30 ␮mol/L bicuculline.29
Brain slices were prepared 24 hours after the ischemic insult.
Whole patch clamp recordings were obtained within 0.5 to 3 hours
after slice preparation. Within this period, the viability of the neurons
within the slice was perfect. In fact, although in the ischemic core no
recording was possible, in the peri-infarct area as well as in the
surrounding normal tissue both spiny neurons and cholinergic
interneurons showed intrinsic and synaptic membrane properties
similar to those previously reported for “healthy” neurons.27–29
Although from each single slice we could obtain several wholecell recordings, we used only one cell to perform the pharmacological experiments with NCX inhibitors to avoid multiple applications
of these drugs on the same brain slice preparation.
Statistical Analysis and Drug Application
Values represent mean⫾SEM of changes in the respective cell
populations. Student t test was used to compare the means. Bepridil
(Sigma, Milan, Italy) was applied by dissolving it to the desired final
concentration in the saline solution. 5-(N-4-chlorobenzyl)-2⬘,4⬘dimethylbenzamil (CB-DMB) was from Division of Pharmacology,
Department of Neuroscience, School of Medicine, “Federico II”
University of Naples, Italy.
Results
Pharmacological Blockade of NCX and Field
Potentials Recorded in the Peri-Infarct Area
As shown in Figure 1A, the peri-infarct area was detected by
morphological and electrophysiological analysis by field
potential detections28 at 24 hours after the ischemic insult.
From sham-operated animals, slices were obtained at the
same time window. Application of bepridil (100 ␮mol/L) in
the extracellular medium for 15 minutes significantly reduced
the amplitude of the field potential obtained 24 hours (n⫽8,
***P⬍0.0001 field potential amplitude t⫽⫺15 versus t⫽40)
after the permanent middle cerebral artery occlusion (Figure
1B and 1C). No significant inhibition was detected in slices
prepared from sham-operated animals at the same timewindow (100 ␮mol/L, n⫽8, P⬎0.05 field potential amplitude
t⫽⫺15 versus t⫽40; Figure 1B and 1C).
As shown in Figure 2, the inhibitory action of bepridil on
the amplitude of field potentials recorded from the periinfarct area of rats exposed to permanent middle cerebral
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Figure 1. The inhibition of the NCX induced by bepridil reduces
the field potential amplitude recorded from the peri-infarct area
in rat corticostriatal slices. A, Electrophysiological and morphological characterization of the peri-infarct area (P) and of the ischemic core (C). B, The plot represents the effect of bath application of 100 ␮mol/L bepridil in corticostriatal slices obtained at
24 hours after permanent middle cerebral artery occlusion in
multiple experiments and in slices obtained from sham-operated
animals. C, Traces represent examples of corticostriatal field
potentials recorded before, during, and after bath application of
100 ␮mol/L bepridil in slices obtained from sham-operated animals (upper row) and from animals exposed to focal ischemia
(lower rows).
Figure 2. Bepridil decreases the field potential amplitude in ischemic slices in a dose-dependent manner (A). The selective
NCX inhibitor CB-DMB mimics the inhibitory effects of bepridil
of field potential amplitude in a dose-dependent manner (B).
The graph shows the dose-response curve obtained from ischemic tissue and from sham-operated striata. Each data point represents at least 3 single experiments. Traces were obtained from
single experiments in ischemic and sham-operated animals (C).
artery occlusion was dose-dependent (n⫽6 for each dose;
IC50⫽68 ␮mol/L).
The inhibitory action of bepridil was also reproduced with
CB-DMB as shown in the dose-response curve of Figure 2
(IC50 of 6 ␮mol/L). This compound, synthesized by the
amiloride discoverer Cragoe,30 is the most potent and selective pyrazine derivative, which inhibits forward and reverse
mode of NCX operation.31–36 Interestingly, this NCX inhibitor, at the incubation time intervals used in the present study,
does not interfere with L-type Ca2⫹ channels.37 However,
more recent putative NCX inhibitors including KB-R7943
Tortiglione et al
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Figure 3. The inhibition of the NCX produces an inward current in striatal medium
spiny neurons (MS), but not in large cholinergic interneurons (LA) recorded from
the peri-infarct area. A, Changes of the
membrane currents detected by wholecell patch clamp recordings in spiny neurons versus cholinergic interneurons in
slices obtained either from sham-operated
animals (upper rows) or from animals
exposed to focal ischemia (lower rows).
The holding potential was kept constant
during all the experiment and it was ⫺80
mV for spiny neurons and ⫺60 mV for
cholinergic interneurons. B, The histogram
shows the percentage of spiny neurons
showing irreversible inward currents in
sham-operated rats and in animals
exposed to ischemia 24 hours before. C,
The plot shows the time-course of the
bepridil-induced inward currents in spiny
neurons recorded from the peri-infarct
area. D, The plot shows an example of a
current–voltage relationship measured
from spiny neurons recorded in the periinfarct area and obtained by delivering
both positive and negative voltage steps
from the holding potential.
and SN-6 can also interfere with several ion transporters and
channels.38 To rule out that the reduction of the field potential
observed in the peri-infarct area after the administration of
NCX inhibitors could be caused by tissue deterioration during
electrophysiological recordings rather than by the specific
drug effect, we performed field potential recordings in the
peri-infarct area also in control medium. Under this experimental condition, we have observed no change of the field
potential amplitude throughout the recording time (n⫽9, data
not shown).
Effect of NCX Inhibitors on Membrane Currents
of Medium Spiny Neurons and Large Aspiny
Interneurons Recorded in the Peri-Infarct Area
To asses the possible mechanism underlying the irreversible
loss of the field potential amplitude caused by bepridil in
spiny neurons after ischemia, we measured, by using wholecell patch clamp recordings, the effects of NCX inhibitors on
somatic currents recorded either in the striatal peri-infarct
area or in the striatum of sham-operated rats. As shown in
Figure 3A through 3C, 100 ␮mol/L bepridil induced an
inward current in most of the medium spiny neurons (8/12)
recorded from the peri-infarct area of striatal slices obtained
24 hours after the ischemic insult (P⬍0.01 15 minutes after
the application of bepridil). Conversely, spiny neurons recorded from sham-operated striata did not show significant
changes of resting membrane currents (11/11) (P⬎0.05, 15
minutes after the application of bepridil). Interestingly, cholinergic interneurons did not show bepridil-induced current
when recorded either from the peri-infarct area (7/7)
(P⬎0.05, 15 minutes after the application of bepridil), or
from control tissue (6/6) (P⬎0.05, 15 minutes after the
application of bepridil). The bepridil-induced inward current
detected in the spiny neurons from the peri-infarct area
developed within 5 minutes from the onset of the application
of bepridil and reached a peak at ⬇15 minutes. This inward
current did not tend to reverse after bepridil removal.
Figure 3D shows a voltage clamp experiment performed by
applying increasing voltage steps in both directions to detect
a reversal potential for the bepridil-induced inward current.
The reversal potential for this current was estimated close to
⫺45 mV (n⫽4). CB-DMB 10 ␮mol/L mimicked the effects
of bepridil, inducing an inward current (at least 50 pA) in 5 of
6 spiny neurons recorded in the peri-infarct area (data not
shown). No current was detected in 5 spiny neurons obtained
from sham-operated rats. Moreover, no current was induced
in cholinergic interneurons recorded either from ischemic
tissue (n⫽4) or from sham-operated animals (n⫽5) (data not
shown).
Effect of NCX Inhibitors on Evoked Excitatory
Synaptic Current Recorded From Medium Spiny
Neurons in the Peri-Infarct Area
Figure 4 shows that application of bepridil causes a progressive reduction of the amplitude of excitatory postsynaptic
currents (EPSCs) recorded from medium spiny neurons in the
peri-infarct area (n⫽7, P⬍0.05). Also in this case, as well as
for the bepridil-induced inward current, the EPSCs did not
recover after the interruption of the drug application. In
medium spiny neurons obtained from the striatum of shamoperated rats, no change of EPSC amplitude was observed
(n⫽7, P⬎0.05).
To investigate whether the depression of EPSCs induced
by bepridil in spiny neurons from the peri-infarct area was
dependent on presynaptic or postsynaptic sites of action, we
measured synaptic responses to a pair of stimuli before and
during the application of the NCX inhibitor. In these experiments, interstimulus interval was 60 ms. Paired-pulse modification of neurotransmission has been studied extensively
and is attributed to a presynaptic change in release probabil-
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Figure 4. Inhibition of the NCX by bepridil
decreases excitatory synaptic currents
measured in spiny neurons recorded from
the peri-infarct area. A, The plot shows
the time course of the EPSC amplitude
detected in spiny neurons obtained either
from sham-operated animals or from rats
exposed to ischemia. The traces in the
lower part of the panel show examples of
EPSCs recorded before and after the
application of bepridil in these 2 experimental conditions. In both the experiments
the holding potential was ⫺80 mV. B, The
histogram represent the EPSC2/EPSC1
ratio in a paired-pulse experiment before
and after the application of 100 ␮mol/L
bepridil.
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ity. An increase in the ratio of the second pulse response to
the first pulse response (EPSC2/EPSC1) indicates a decrease
in the release probability. The depression of the EPSC
amplitude induced by 100 ␮mol/L bepridil was not associated
with a significant increase in this ratio (n⫽5, P⬎0.05; Figure
4B), suggesting that postsynaptic mechanisms play a role in
the observed effect.
The synaptic effects of bepridil were mimicked by
10 ␮mol/L CB-DMB (n⫽7, data not shown).
Discussion
The present study represents the first electrophysiological
demonstration that the activation of NCX maintains ionic
homeostasis in the peri-infarct area after focal ischemia. We
found, in fact, that the pharmacological inhibition of NCX by
bepridil and CB-DMB causes an irreversible loss of field
potential amplitude recorded from the striatal area surrounding the ischemic core. These effects were dose-related and
irreversible suggesting that inhibition of NCX can trigger a
cascade of detrimental events leading to neuronal damage.
The hypothesis that the electrophysiological action of bepridil
and CB-DMB are closely linked to the ischemia-triggered
events is also confirmed by the evidence that these drugs
failed to alter the field potential amplitude in slices obtained
from sham-operated rats.
Bepridil is not considered a selective NCX inhibitor. In
fact, this drug inhibits sodium and calcium channels and
modulates potassium conductances. Nevertheless, the use of a
more specific NCX inhibitor, such as CB-DMB,18,31,33,34
mimicking the electrophysiological effects of bepridil
strongly support the hypothesis that the effects we observed
are dependent on the selective inhibition of NCX. The change
of the expression in NCX1, NCX2, and NCX3 gene products
after permanent middle cerebral artery occlusion seems to
suggest that these isoforms can be involved in the process
leading to cell death or survival in those areas that are
interested by the ischemic insult.6,7,9
According to our electrophysiological and pharmacological data, it has been shown that after knocking down 2 of
these proteins, NCX1 and NCX3, a remarkable enlargement
of the infarct volume occurred. Such a phenomenon was
associated with a worsening of neurological deficits.7 Accordingly, the pharmacological inhibition of NCX leads to an
enlargement of the infarct size.6
By using single-cell patch clamp recordings, we have
demonstrated that the pharmacological blockade of NCX
produced an inward current in spiny neurons recorded in the
peri-infarct area, whereas no current was detected in cells
obtained from sham-operated animals. The estimated reversal
potential for this inward current was ⬇⫺45 mV, indicating
that multiple events rather than a single conductance seem to
be involved.27 The single cell experiments gave us also the
possibility to demonstrate that the pharmacological action of
bepridil and CB-DMB were cell-type–specific. In fact, no
inward current was detected from striatal large cholinergic
interneurons recorded from the peri-infarct area. Noticeably,
striatal spiny neurons are vulnerable to energy deprivation,4,20,21,29 whereas cholinergic interneurons have been reported to be resistant to ischemia.4,5,20,21 Thus, we can argue
that the different expression of NCX among various striatal
neuronal subtypes might take part in the mechanisms controlling cell-type–specific vulnerability to ischemia within the
striatum and, possibly, in other brain areas. However, as an
alternative hypothesis, we can consider the possibility that
although the expression of NCX is similar in spiny neurons
and cholinergic interneurons, these 2 neuronal subtypes
respond in a distinct manner to NCX inhibition because they
have a differential capability to compensate alterations of ion
concentrations.
The inward current recorded from spiny neurons during
application of NCX inhibitors was paralleled by a progressive
Tortiglione et al
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decrease of the amplitude of the evoked EPSCs. This latter
effect may also represent the synaptic event underlying loss
of the field potential amplitude detected by extracellular
recordings. In fact, the field potential amplitude reflects the
synchronous discharge of several neurons adjacent to the
recording electrode and activated by excitatory fibers. Interestingly, the reduction of the EPSC amplitude was not
coupled to changes in EPSC2/EPSC1 ratio, suggesting that
postsynaptic rather than presynaptic mechanisms seem to
underlie this event.
An interesting issue that should be considered in future
studies is related to the potential recovery of the peri-infarct
area and its relationship with the functional activity of NCX
at different time windows after the ischemic insult.
The 3 isoforms of NCX that are present in the striatum are
constitutively expressed not only in neurons but also in glia.39
Interestingly, the expression of this antiporter can be altered
under particular conditions, such as hypoxia.9
Cerebral edema is known to exacerbate neuronal damage
induced by ischemia. Accordingly, it has been recently
reported that a nonselective cation channel is expressed after
brain ischemia and is crucially involved in development of
cerebral edema.40 However, no evidence has never been
provided on an involvement of NCX on water distribution
either in normal tissue or in focal ischemia.
NCX is also expressed in peripheral cells such as macrophages and leukocytes1 migrating in the brain after the
ischemic insult; therefore, a hypothetical role played by the
modification of NCX in these cells cannot be excluded.
Nevertheless, the changes observed in the electrophysiological behavior of spiny and cholinergic interneurons seem to
favor the idea that the action of the NCX inhibitors is mainly
directed to neuronal cells rather than in peripheral cells.
In conclusion, our data suggest that in the peri-infarct area,
NCX plays a role in maintaining ionic homostasis because its
pharmacological block leads to detrimental effects after focal
cerebral ischemia. Moreover, this effect is selectively expressed
in neurons particularly vulnerable to energy deprivation.
Acknowledgments
We thank Mr Cristiano Spaccatini and Mr Massimo Tolu for their
excellent technical support.
Sources of Funding
This work was supported by grants from Ministero della Salute
(Ricerca Finalizzata 2004, 2005 IRCCS to P.C. and B.P.), FIRB
2001, Fondazione Cassa Risparmio Perugia (to P.C.).
Disclosures
None.
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Na+/Ca2+ Exchanger Maintains Ionic Homeostasis in the Peri-Infarct Area
Anna Tortiglione, Barbara Picconi, Ilaria Barone, Diego Centonze, Silvia Rossi, Cinzia Costa,
Massimiliano Di Filippo, Alessandro Tozzi, Michela Tantucci, Giorgio Bernardi, Lucio
Annunziato and Paolo Calabresi
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Stroke. 2007;38:1614-1620; originally published online March 29, 2007;
doi: 10.1161/STROKEAHA.106.478644
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